CN105671133A - Human body BMR (Basal Metabolic Rate) detection kit and calculation method - Google Patents

Abstract

The invention provides a human body BMR (Basal Metabolic Rate) gene detection kit, which comprises a kit body and reagents singly stored in the kit body, wherein the reagents comprise (1) PCR (Polymerase Chain Reaction) primer groups aiming at SNP (Single Nucleotide Polymorphism) polymorphic sites of two genes; (2) a PCR amplification reagent; and (3) an agarose gel electrophoresis analysis reagent. Through the PCR primer design, a plurality of PCR amplification reactions can be synchronously performed on the same PCR instrument; relevant genes of a testee are detected; the effectiveness and the specificity of the detection are realized; the genetic factor of the BMR of the testee can be obtained through analysis, so that a proper health scheme is provided for the testee; the testee can know the self metaboilic level; the guidance can be realized on the scientific and reasonable exercise intensity and daily diet management; and the corresponding strategies are used for avoiding methods which are noneffective or even harmful for the testee in the body building exercise taking and fat loosing processes.

Description

Human body BMR(basal metabolic rate) detection kit and computational methods

Technical field

The present invention relates to gene test, be specifically related to human body BMR gene detecting kit.

Background technology

Basal metabolic rate refers in natural temperature environment, and human body (includes digestive system, i.e. fasting more than two hours) under inactive state, and sustain life the required minimum energy consumed. These energy are mainly used in keeping the function of each organ, drain (kidney), removing toxic substances (liver), musculation etc. as breathed (lung), heart beating (heart), glandular secretion (brain and other nervous system), filtering. Basal metabolic rate along with the age increases or loses weight and reduces, and can increase along with muscle and increase. Disease, feed, variation of ambient temperature, bear pressure level change and all can change the energy expenditure of human body, thus affecting basal metabolic rate.

The heat that basal metabolic rate difference determines human body consumption every day is different, if therefore to be lost weight by diet, then must take in energy < consumed energy. Meanwhile, basal metabolic rate also determines human body catabiotic difference under different exercise intensitys, therefore in the middle of body building process, if intensity is excessive causes that energy expenditure is too much, can cause many unhealthy even harmful sufferers.

BMR is the index of each organ of human body, tissue energy consumption, therefore closely related with gene. The computational methods of current various BMR, mainly by the method for statistics, are undertaken by corresponding instrument, it is impossible to the difference between reaction individuality.

Summary of the invention

In view of this, the invention provides a kind of BMR gene detecting kit, the related gene of testee is detected by this test kit, analysis obtains its internal metabolism level, and calculate the BMR numerical value based on gene information, and pcr amplification reaction is synchronously performed in same PCR instrument and possesses the high efficiency of detection, specificity.

A kind of BMR gene detecting kit, including the reagent individually deposited in box body and box body, the reagent deposited includes:

(1) primer sets is reacted for the PCR of 2 gene SNP polymorphic sites:

Primer sets for SNPRS16892496:

5’-GTTGAAGAGCAAGCCCCCA-3’

5’-GTGACTTGTACCCACG-3’

5’-GAACCAATAATACTCTCCTC-3’

5’-CATTTGGAATGGAAAC-3’

Primer sets for SNPRS7832552:

5’-GTGATAGTGTGAGGTAC-3’

5’-GTTTATTAAGAGCATTCA-3’

5’-ATCTTTAAAGAATTCTAG-3’

5’-TGCATTGGATTTTG-3’

(2) pcr amplification reagent;

(3) agarose gel electrophoresis analytical reagent.

BMR gene detecting kit provided by the invention provides the benefit that: make multiple pcr amplification reaction be synchronously performed in same PCR instrument by the design of PCR primer, the related gene of testee is detected, and realize the high efficiency of detection, specificity, analyze the inherited genetic factors obtaining its BMR, thus obtaining a suitable healthy scheme for testee, understand the metaboilic level of self, instruct its scientific and reasonable exercise intensity and diet management, take corresponding strategy to avoid the method that it is invalid or even harmful in body building and fat-reducing.

Accompanying drawing explanation

Fig. 1 is product agarose gel electrophoresis figure.

Detailed description of the invention

BMR gene detecting kit, including the reagent individually deposited in box body and box body, the reagent deposited includes:

(1) primer sets is reacted for the PCR of 2 gene SNP polymorphic sites:

Primer sets for SNPRS16892496:

5’-GTTGAAGAGCAAGCCCCCA-3’

5’-GTGACTTGTACCCACG-3’

5’-GAACCAATAATACTCTCCTC-3’

5’-CATTTGGAATGGAAAC-3’

Primer sets for SNPRS7832552:

5’-GTGATAGTGTGAGGTAC-3’

5’-GTTTATTAAGAGCATTCA-3’

5’-ATCTTTAAAGAATTCTAG-3’

5’-TGCATTGGATTTTG-3’

(2) pcr amplification reagent;

(3) agarose gel electrophoresis analytical reagent.

Below in conjunction with embodiment, method provided by the invention is further described.

Tester's basal conditions is: male, height 175cm, body weight 100 kilograms. Tester is adopted BMR gene detecting kit to detect the SNP polymorphic site of 2 genes by the present embodiment simultaneously, and according to genotypic results, analyze the inherited genetic factors obtaining its BMR, thus obtaining a suitable healthy scheme for testee, understand the metaboilic level of self, instruct its scientific and reasonable exercise intensity and diet management, take corresponding strategy to avoid the method that it is invalid or even harmful in body building and fat-reducing.

In the BMR gene detecting kit that use is arrived, reagent is by consisting of:

(1) primer sets is reacted for the PCR of 2 gene SNP polymorphic sites:

Primer sets for SNPRS16892496:

5’-GTTGAAGAGCAAGCCCCCA-3’

5’-GTGACTTGTACCCACG-3’

5’-GAACCAATAATACTCTCCTC-3’

5’-CATTTGGAATGGAAAC-3’

Primer sets for SNPRS7832552:

5’-GTGATAGTGTGAGGTAC-3’

5’-GTTTATTAAGAGCATTCA-3’

5’-ATCTTTAAAGAATTCTAG-3’

5’-TGCATTGGATTTTG-3’

(2) pcr amplification reagent: 10 × PCR buffer, this PCR buffer is 100mMTris-HClpH8.3,500mMKCl, 15mMMgCl2; DNTPs mixture, this dNTPs mixture is triphosphoric acid guanine deoxyribonucleoside acid dGTP, triphosphoric acid adenyl-deoxyribonucleotide dATP, triphosphoric acid thymidylic acid dTTP, tetra-kinds of nucleotide of triphosphoric acid deoxycytidylic acid dCTP, each concentration of component is 2.5mM; 5U/ μ l hot start Taq polymerase.

(3) agarose gel electrophoresis analytical reagent: agarose, TAE buffer, DNA molecular amount standard, Goldview dyeing liquor and DNA sample-loading buffer, this buffer with bromophenol blue for indicator dilution to 1X after use.

Above-mentioned BMR gene detecting kit is used to detect as follows:

(1) sample genomic dna is extracted.

Gather this tester's saliva, in 1-2ml saliva sample, add 500ul extract buffer solution, this DNA extraction buffer solution solvent is the NaCl of EDTA and the 50mM of Tris-HClpH7.4,0.5mM of 50mM, repeatedly after piping and druming mixing, centrifugal 5 minutes of 8000 × g, supernatant discarded, this step is repeated once; The precipitation obtained adds 500ul lysate, this lysate solvent is 50mMTris-HClpH7.4,50mMTris-HClpH7.4,150mMNaCl, 1mMEDTA, 1%Tritonx-100,1%Sodiumdeoxycholate, 0.1%SDS, E.C. 3.4.21.64 20mg/mL, after the precipitation that thoroughly suspends fully mixing, room temperature is placed 30 minutes, and period reverse centrifuge tube back and forth is for several times;In the mixed liquor obtained, add the aqueous solution 10 μ L that concentration is 10mg/mLRNA enzyme, at 37 DEG C, stand 10min; In the supernatant obtained, adding equal-volume phenol chloroform mixed solution, phenol and chloroform volume ratio are 1: 1, fully mix, mixed liquor 4 DEG C, centrifugal 5 minutes of 12000 × g, and supernatant moves in clean centrifuge tube; Adding equal-volume benzene atmosphere-chloroform-isoamyl alcohol mixed solution, phenol, chloroform and isoamyl alcohol volume ratio are 25: 24: 1, fully after mixing, and 4 DEG C, centrifugal 5 minutes of 12000 × g, supernatant moves in clean centrifuge tube; Add equal-volume chloroform-isoamyl alcohol mixed solution, fully after mixing, 4 DEG C, centrifugal 5 minutes of 12000 × g, supernatant moves in clean centrifuge tube; Adding the ice bath aqueous isopropanol of 0.6 times of volume and the 3mol/L sodium acetate solution of 0.1 times ,-20 DEG C of standings are after 60 minutes, and 4 DEG C, 12000 × g is centrifuged 10 minutes, abandons supernatant; The precipitate obtained adds 0.5mL70% ethanol purge precipitate, 4 DEG C, centrifugal 5 minutes of 12000 × g, abandon supernatant, this step is repeated once; The precipitate natural air drying obtained, adds the 20 aseptic ultra-pure water back dissolvings of μ L, electrophoresis detection ,-20 DEG C of preservations.

(2) pcr amplification

The present embodiment detects RS16892496 and RS7832552 simultaneously. The detection of each SNP needs a pair upstream and downstream primer, two polymorphic primer totally 4 PCR primer, needs 8 primers altogether. The detection of each SNP needs to do two pipe pcr amplifications, for preventing the reaction of false positive etc, whether successful weighs PCR, add a pipe negative control group, being 5 pipe PCR altogether, described negative control group genomic templates and reaction system are the same, but do not have primer.

According to different detection site, prepare PCR reaction system with corresponding primer respectively in following ratio: 2.5 μ l10 × PCR buffer, 2 μ ldNTPs, 0.5 μ l primer sets, hot start Taq polymerase 0.15 μ l, genomic templates 80mg, adding ultra-pure water to cumulative volume is 25 μ l.

Will be equipped with the Eppendorf test tube of reaction solution to put in ABI9700PCR instrument, it is as follows that reaction condition is set: 95 DEG C of 3min of denaturation; 94 DEG C of 30s of thermal cycle, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations; Extend 72 DEG C of 3min. After reaction terminates, take out test tube.

Owing to 5 pipe pcr amplification reactions are synchronously performed in same PCR instrument, in order to realize the high efficiency of detection, specificity, require that the Tm value of 8 PCR primer is close, need for this first to carry out substantial amounts of DNA sequence software analysis to design PCR primer, and carry out the optimization of Tm value by substantial amounts of experiment and determine the reasonability of design, the detection of this test kit each gene SNP polymorphism is not only relatively independent but also connect each other, indivisible.

(3) agarose gel electrophoresis detection

Product after amplification is carried out sepharose electrophoresis detection, and process is as follows: 3g agarose, adds 100mL deionized water, microwave-oven-heating 1min, adds 5 μ LGoldview dyeing liquors, make the agarose gel of 3% when being cooled to 60 DEG C. PCR primer 10 μ L and 6 × tbe buffer liquid 0.8 μ L mixes loading, voltage 100V electrophoresis 15min. Reading result under uviol lamp and take pictures, gained sepharose electrophoresis detection figure is as shown in Figure 1.

It is as follows that detection obtains 2 gene types analysis results:

SNP	Genotyping result
		RS16892496	TT
RS7832552	TT

According to above genotyping result, the contribution margin (Y) of the gene pairs basal metabolic rate of this tester is: 2.7+2.55=5.25KG.According to being RS16892496GG > the LBM 2.55KG more than the LBM of TC and CC type than many 2.7KG, the RS7832552TT type of TT and TG type of TG/TTGG type. Then according to below equation:

Male: LBM=(0.32810*W)+(0.33929*H)-29.5336+Y

Women: LBM=(0.29569*W)+(0.41813*H)-43.2933+Y

BMR=P(kcal/day)=370+(21.6*LBM)

This tester is male, therefore calculates its BMR=1837kcal/day. Energy expenditure in its daily life can be calculated according to following table:

Seldom or not move Dailykilocaloriesneeded=BMRx1.2

More slight exercise (1 3 days/weekly) Dailykilocaloriesneeded=BMRx1.375

Moderate exercise (3 5 days/weekly) Dailykilocaloriesneeded=BMRx1.55

Severe exercise (6 7 days/weekly) Dailykilocaloriesneeded=BMRx1.725

Pole severe tempers (twice daily) Dailykilocaloriesneeded=BMRx1.9

Be engaged in more slight exercise for as daily in this man, then the energy expenditure of its every day is approximately: 1723*1.375=2525kcal.

The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all within the spirit and principles in the present invention, any amendment of making, equivalent replacement, improvement etc., should be included within protection scope of the present invention.

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Claims (10)

1. basal metabolic rate gene detecting kit, including the reagent individually deposited in box body and box body, it is characterised in that: the reagent deposited includes:

(1) primer sets is reacted for the PCR of 2 gene SNP polymorphic sites:

Primer sets for SNPRS16892496:

5’-GTTGAAGAGCAAGCCCCCA-3’

5’-GTGACTTGTACCCACG-3’

5’-GAACCAATAATACTCTCCTC-3’

5’-CATTTGGAATGGAAAC-3’

Primer sets for SNPRS7832552:

5’-GTGATAGTGTGAGGTAC-3’

5’-GTTTATTAAGAGCATTCA-3’

5’-ATCTTTAAAGAATTCTAG-3’

5’-TGCATTGGATTTTG-3’

(2) pcr amplification reagent;

(3) agarose gel electrophoresis analytical reagent.

2. basal metabolic rate gene detecting kit according to claim 1, it is characterised in that: described primer consumption is 0.5-1.0 μ l.

3. basal metabolic rate gene detecting kit according to claim 1, it is characterised in that: described pcr amplification reagent includes: 10 × PCR buffer, dNTPs, hot start Taq polymerase, genomic templates.

4. basal metabolic rate gene detecting kit according to claim 3, it is characterised in that: described PCR buffer comprises: 100mMTris-HClpH8.3,500mMKCl, 15mMMgCl₂。

5. basal metabolic rate gene detecting kit according to claim 3, it is characterised in that: described pcr amplification reagent dosage is: 10 × PCR buffer 2.5 μ l, dNTPs2 μ l, hot start Taq polymerase 0.1-0.2 μ l, genomic templates 50-100ng.

6. basal metabolic rate gene detecting kit according to claim 1, it is characterised in that: described agarose gel electrophoresis analytical reagent includes: agarose, TAE buffer, DNA molecular amount standard, DNA non-toxic dye and DNA sample-loading buffer.

7. basal metabolic rate gene detecting kit according to claim 6, it is characterised in that: the preferred BiotiumGelred non-toxic dye of described DNA non-toxic dye.

8. basal metabolic rate gene detecting kit according to claim 1, it is characterized in that: being first depending on genotypic results, to calculate LBM related gene be each gene pairs basal metabolic rate contribution margin sum to total contribution margin of basal metabolic rate, and its unit is kilogram.

9. computational methods according to claim 8, it is characterised in that: the contribution margin of each gene pairs body weight is determine according to the concrete outcome of typing.

10., according to claim 1,8 and 9, calculate BMR according to formula, it is characterised in that these computational methods take into account the impact of gene.